Valvular interstitial cells (VICs) play a vital role in the function of the normal heart valve by maintaining the structural integrity of the leaflets. However, excessive activation of VICs to the highly synthetic and contractile myofibroblast phenotype is associated with valve pathologies that results in thousands of heart valve replacements annually. Our long term goal is to elucidate the mechanisms of mechanical and biochemical regulation of VIC activation to facilitate the development of treatments for valve fibrosis including tissue engineered valves. Towards this goal, the objective of this proposal is to determine the role of boundary stiffness on the activation of VICs, particularly in response to cytokines such as transforming growth factor beta1 (TGF-beta1), endothelin-1 (ET-1), and fibroblast growth factor 2 (FGF2). Our hypothesis that boundary stiffness modulates the force which VICs generate in compliant extracellular matrix, and this intrinsic tension in turn regulates cytokine-induced VIC activation to the myofibroblast phenotype has several important implications not previously considered. Specifically, 1) stiff areas of the valve may serve to potentiate fibrosis in adjacent healthy areas by increasing the cells'sensitivity to profibrotic cytokines in these compliant regions, and 2) low boundary stiffness may spare compliant regions from fibrosis and reducing boundary stiffness may reverse fibrosis. Specific Aim 1 will examine the relationships between VIC activation and boundary stiffness. VICs will be cultured within a novel compliant anchored collagen gel system which precisely controls boundary stiffness. Myofibroblast activation will be characterized by functional measures including contractile force and matrix remodeling and confirmed by alpha smooth muscle actin (ASMA) and SMemb (a nonmuscle myosin) expression. Specific Aim 2 will serve to characterize the combined effects of boundary stiffness and cytokines. VICs will be cultured and analyzed as in SA1 and also stimulated with pro- and anti-fibrotic cytokines (TGF- beta1, ET-1, FGF2). Quantitative dose-response relationships including interactions among factors will be determined utilizing design of experiment (DOE) methods. Specific Aim 3 will determine the role of reduced boundary stiffness level on the persistence of myofibroblast activity. Activated VICs will be cultured in rigidly anchored collagen gels;the boundary stiffness will then be decreased to predetermined levels. Changes in VIC activation and apoptosis levels will be assessed. Relevance to public health: This research effort strives to broaden our understanding of the causes of heart valve disease, a significant public health concern in the United States. The knowledge gained from this proposal will aid in the development of treatments for diseased heart valves including minimally invasive procedures and tissue engineered replacement valves. PUBLIC HEALTH RELEVANCE: The goal of this study is to determine how regional mechanical and chemical cues interact to guide the behavior of the cells within native and engineered heart valves. The knowledge gained from the proposed studies will aid in the development of minimally invasive treatments for heart valve disease and facilitate development of tissue engineered valves.